The Na+:H+ exchangers (NHE) are present in both apical and basolateral membranes of the medullary thick ascending limb (MTAL). Recent studies suggest that, during antidiuresis, apical NHE is inhibited while basolateral NHE is activated. This novel discordant regulation of the apical and basolateral NHE is critical for the survival of MTAL and its function to facilitate regulating acid/base during antidiuresis. The mechanism, however, is not clear. This proposal will study the mechanisms by which acute and chronic antidiuresis regulate apical and basolateral NHE in the mouse MTAL using the strategy that the effects of antidiuresis are mediated by vasopressin and hyperosmolality. For acute antidiuresis, I will study the acute effects of AVP, hyperosmolality and their interactions on the apical and basolateral NHE in in vitro perfused mouse MTAL tubules. Previous studies indicate that AVP-stimulated ion transport in the MTAL is mediated via V2 receptor/cAMPprotein kinase (PKA) pathway. In addition, my preliminary data indicate that the effects of AVP and hyperosmolality on the NHE in this tubule segment are mediated via different mechanisms. These results lead to my hypothesis that AVP, but not hyperosmolality, regulates NHE in the MTAL via cAMP/PKA pathway. To test this hypothesis, I will examine in the mouse MTAL whether the effects of these two factors on the apical and basolateral NHE can be mimicked by agonists of cAMP/PKA pathway and abolished by the antagonists of this pathway. To confirm the results, I will identify NHE isoforms in the apical and basolateral membranes of the mouse MTAL using molecular techniques. Then, I will study the effects of these two factors on the phosphorylation of NHE isoforms and examine whether the phosphorylation is mediated via cAMP/PKA pathway. For chronic antidiuresis, I will assess the effects of chronic water deprivation on the functional expression of the apical and basolateral NHE and molecular (mRNA and protein) expression of the NHE isoforms in these membranes in the mouse MTAL. My preliminary data show that the expression of basolateral NHE isoforms during chronic antidiuresis differs from that under acute antidiuresis, which has important physiological implications for the chronic adaptation of MTAL to the hyperosmotic environment. It is difficult to study phosphorylation of NHE in the native MTAL cells and is impossible to delineate whether the effects of chronic antidiuresis on the NHE are mediated via AVP and/or hyperosmolality. Therefore, to address these two issues, I have developed a mouse MTAL cell line containing NHE with similar properties as those in the native MTAL cells. The results of this grant proposal will provide important insights into adaptation of MTAL and other renal medullary cells to hyperosmolality in vivo and into maintaining net acid excretion during antidiuresis in physiological and pathological states.